Hopanoid composition mediates bacterial ethanol tolerance

Abstract Hopanoids are abundant membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The fermenter Zymomonas mobilis features the highest measured concentration of hopanoids - up to 50% of all lipids in the cell1 - leading to the hypothesis that these lipids can protect against ethanol toxicity. In principle, the condensing effect of hopanoids, analogous to eukaryotic sterols2,3, could buffer against changes to membrane structure induced by solvents. However, there is no clear link between ethanol exposure and hopanoid biosynthesis in Z. mobilis1,4,5, and the lack of genetic tools for manipulating hopanoid composition in vivo has limited their further functional analysis. Because of polyploidy (> 50 genome copies per cell), we found that disruptions of essential hopanoid biosynthesis (hpn) genes in Z. mobilis act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrated that both reduced hopanoid content and modified hopanoid head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their composition, contributes to fitness at low pH. Spectroscopic analysis of model membranes showed that hopanoids protect against several ethanol-driven phase transitions in bilayer structure, including bilayer expansion and lipid interdigitation. We propose a model by which hopanoids act through a combination of hydrophobic and inter-lipid hydrogen bonding interactions to stabilize bacterial membranes against chemical stress.